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. 2018 Jun 5;8(11):e2877.
doi: 10.21769/BioProtoc.2877.

Expansion of Airway Basal Cells and Generation of Polarized Epithelium

Hannah Levardon  1   2   3 , Lael M Yonker  3   4   5 , Bryan P Hurley  3   4 , Hongmei Mou  3   4   5   6
Affiliations

Expansion of Airway Basal Cells and Generation of Polarized Epithelium

Hannah Levardon et al. Bio Protoc. .

Abstract

Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies. Further, basal stem cells can be harvested from patients with a specific airway disease, such as cystic fibrosis, enabling investigation of potentially altered behavior of disease-specific cells in the appropriate context of the airway mucosa. Here we describe, in detail, a protocol for the serial expansion of airway basal stem cells to enable the generation of nearly unlimited airway basal cells that can be stored and readily available for subsequent culturing and differentiation. In addition, we describe culturing and differentiation of airway basal stem cells on permeable transwell filters at air-liquid interface to create functional mucociliary pseudostratified polarized airway epithelial mucosa.

Keywords: Airway basal stem cells; Airway diseases modeling; BMP/TGFβ/SMAD signaling; Mucociliary differentiation on air-liquid interface (ALI); Pseudostratified airway epithelium; Stem cell expansion.

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Figures

Figure 1.
Figure 1.. Serial expansion and immunofluorescence characterization of human airway basal cells.
A. Schematic of serial expansion of human airway basal cells. B. Phase contrast images of the human basal cells at various days after cell seeding (initial seeding density is 10-15%). Scale bars = 50 μm. C. Immunofluorescence of basal stem cell markers (CK5 and p63) and airway-specific transcription factors (NKX2.1 and FOXA2) on cultured airway basal cells (Passage 6). Scale bars = 20 μm.
Figure 2.
Figure 2.. Mucociliary differentiation of human airway basal cells cultured on transwell membrane in airway-liquid interface.
A. Schematic description of Transwell® permeable supports used for differentiation of airway basal cells; B. Schematic strategy of air-liquid interface differentiation; C. The phase contrast images of primary airway epithelial culture of ALI culture at variable time points.
Video 1.
Video 1.. Human airway stem cells (P6) were differentiated on ALI for 20 days.
The cilia beating was visualized under the Olympus IX81 inverted microscope. Scale bar = 20 μm.
Figure 3.
Figure 3.. Immunofluorescence characterization of human airway basal cells cultured on airway-liquid interface.
A. Immunofluorescence for markers of differentiation (acetylated-tubulin for ciliated cells, CC10 for club cells and MUC5AC for goblet cells) using wholemount staining. Scale bars = 50 μm. B. Immunofluorescence for airway epithelial cell markers (acetylated-tubulin and FOXJ1 for ciliated cells, CC10 for club cells and MUC5AC for goblet cells, and p63 for basal stem cells) on transverse ALI membrane sections. Scale bars = 20 μm.
See this image and copyright information in PMC

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